There are several important intracellular signaling proteins in brain tissue that have been linked to regulation of neurotransmitters that are involved in neurological diseases such as depression, schizophrenia, and Parkinson's disease. These intracellular signaling proteins include DARPP-32 (Dopamine- and cAMP-regulated phosphoprotein, Mr 32,000), ERK1 and ERK2 (extracellular signal-regulated protein kinases 1 and 2), and CREB cAMP-response element binding protein).
2.1. DARPP-32
DARPP-32 was discovered as a major target for dopamine (DA) and cAMP in the brain (Walaas et al. 1983. Nature 301:69-71). DARPP-32 is enriched in the two major projection areas for dopaminergic neurons, the prefrontal cortex and the striatum. DARPP-32 plays an obligatory role in the biochemical, electrophysiological, transcriptional, and behavioral effects of dopamine (Greengard, P. et al. 1999. Neuron 23:435-447). One study has linked the level of DARPP-32 with the pharmacological activity of certain anti-depressant compounds (Guitart, X. and E. J. Nestler. 1992. J. Neurochem. 59:1164-1167). These researchers demonstrated that chronic administration of lithium, imipramine, and tranylcypromine in rats produced significant increases in frontal cortex levels of DARPP-32 immunoreactivity, while administration of haloperidol, morphine, and cocaine were without effects on DARPP-32 immunoreactivity. Lithium is used for treatment of manic-depressive illness, while imipramine and tranylcypromine are anti-depressants. Imipramine acts by inhibiting norepinephrine re-uptake while tranylcypromine is a monoamine oxidase inhibitor.
2.2. cAMP Response Element Binding Protein (CREB)
In neurons, Ca2+ influx through different calcium channels activates distinct signaling pathways that either target the serum response element (SRE) or the calcium response element (“CaRE” or “CRE”) within the c-fos promoter (Ghosh et al., J. Neurobiol March 1994;25(3):294-303). Transcription through the CRE requires the induced phosphorylation of the cAMP response element binding protein (CREB) at Ser133. CREB contains basic domain/leucine zipper motifs and binds as a dimer to CRE (De Cesare et al., Prog Nucleic Acid Res Mol Biol 2000;64:343-69). The activation function of CRE-binding proteins such as CREB is modulated by phosphorylation by several kinases and is mediated by coactivators such as CBP and p300 (De Cesare et al., Prog Nucleic Acid Res Mol Biol 2000;64:343-69)
Ca2+ thus regulates gene expression by multiple signaling pathways, including the one that involves the Ca(2+)-dependent phosphorylation of the transcription factor CREB (Ghosh et al., J Neurobiol March 1994;25(3):294-303). CREB is involved in the formation of memory in diverse organisms and regulates the formation of memories of various types of tasks that utilize different brain structures, including long-term memory (LTM) consolidation (Lamprecht, Cell Mol Life Sci April 1999;55(4):554-63; Huang et al., Essays Biochem 1998;33:165-78).
2.3. ERK1 and ERK2
Extracellular signal-regulated kinase 1 (ERK1) and Extracellular signal-regulated kinase 2 (ERK2) are part of the mitogen-activated protein kinase (MAP kinase, MAPK) superfamily. The MAPK superfamily of signaling cascades is a critical regulator of cell division and differentiation (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10) and is also involved in learning and memory (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10). The MAPK cascade is part of a family of signaling cascades that share the motif of three serially linked kinases regulating each other by sequential phosphorylation (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10). The superfamily of MAPK signaling cascades includes the extracellular signal-regulated kinases (ERKs), the JNKs and the p38 stress activated protein kinases (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10).
The most abundant ERKs in the brain are p44 MAPK (ERK1) and p42 MAPK (ERK2). ERK1 and ERK2 serve as intermediates that regulate serine/threonine phosphorylations in downstream intracellular signaling events (see, e.g., Boulton et al., 1991, Cell 65(4):663-75). ERKs are activated through phosphorylation at two sites, Thr202 and Tyr204 (for ERK1) and Thr185 and Tyr 187 (for ERK2).
ERKs are also abundantly expressed in neurons in the mature central nervous system, where the ERK signaling system has been apparently co-opted in mature neurons to function in synaptic plasticity and memory (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10). ERKs also appear to serve as biochemical signal integrators and molecular coincidence detectors for coordinating responses to extracellular signals in neurons (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10). ERK1 and ERK2 are involved in the induction of c-fos via phosphorylation and activation of CREB (Sweatt, 2001, J. Neurochem. (January) 76(1):1-10).
2.4. Classification of Anti-Psychotic Drugs
Anti-psychotic drugs have been classified into two classes: typical and atypical. Typical anti-psychotics include haloperidol, a drug that has been in use for over 30 years. The efficacy of the typical anti-psychotics is limited, however, and their side effects often limit their use as well. Such side effects include acute extrapyramidal effects such as acute dystonias (abnormal muscle spasms and postures), pseudoparkinsonism, and akathisia. Typical anti-psychotics appear to produce their pharmacological effects at least in part through acting as antagonists at the dopamine D2 receptor (see, e.g., Silverstone T., Acta Psychiatr Scand Suppl 1990;358:88-91).
Atypical anti-psychotics were developed in more recent years to overcome some of the limitations of the older typical anti-psychotic compounds. One such drug is clozapine, which is an improvement over drugs such as haloperidol, because it is almost devoid of extrapyramidal side effects. The mechanism of action of atypical anti-psychotics, however, is not well understood. The pharmaceutical industry has tried to develop other drugs with activity similar to clozapine, but development efforts have been hindered by the lack of understanding of the mechanism of action of such typical anti-psychotics.
Anti-psychotic drugs are typically tested in animal models. However, no one model is predictive of clinical efficacy in controlling “positive” psychotic symptoms such as delusions and auditory hallucinations and “negative” symptoms such as flat affect and avolition. In recent years, “atypical” antipsychotics have come into wide use, predominantly because they provide the advantage of greatly reduced or nearly absent extrapyramidal side effects such as pseudoparkinsonism and other movement disorders. One such atypical antipsychotic, clozapine, has been shown to improve negative symptoms and is almost devoid of extrapyramidal side effects. Regular users of clozapine, however, are at risk for agranulocytosis, a lethal rupture of blood cells, and their white blood count must be continuously monitored via expensive laboratory tests. This adds considerably to the cost and limits the availability of this treatment.
Therefore, considerable effort has been expended to find an improved compound with similar antipsychotic properties. Hampered by the relative lack of insight into the mechanism of action of these drugs, investigators attempt to match candidate antipsychotics to clozapine using many different behavioural and biochemical parameters. For example, Millan et al. (2000, J Pharmacology and Experimental Therapeutics 292, 54-66) disclose the use of 14 different animal models and nine different receptor-binding assays to compare prospective antipsychotic compound S18327 (1-{2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)piperid-1-yl]ethyl}3-phenyl imidazolin-2-one) to clozapine.
Another test used to differentiate anti-psychotic drugs is comparison of their abilities to induce immediate-early gene c-fos. Typical and atypical anti-psychotics differently affect the expression of the immediate-early gene c-fos in the dorsal striatum, a region of the brain implicated in controlling movement (Robertson, G. S. et al. 1994. J. Pharmacol. Exp. Ther. 271:1058-1066). Haloperidol, a typical anti-psychotic, is much more effective at inducing c-fos expression as compared to clozapine, an atypical anti-psychotic (Robertson, G. S. et al. 1994. J. Pharmacol. Exp. Ther. 271:1058-1066). Studies have indicated that two mitogen-activated protein kinases, ERK1 and ERK2, are involved in the induction of c-fos via phosphorylation and activation of the transcription factor CREB (Sweatt, J. D. 2001. J. Neurochem. 76:1-10). Clozapine, on the other hand, has weak c-fos inductive activity in the dorsal striatum, but strongly induces c-fos expression in the medial prefrontal cortex. This action may be linked to the ability of clozapine to relieve negative psychotic symptoms. Once again, however, the mechanism underlying this induction is unclear.
Wettstein et al. (1999, Prog Neuropsychopharmacol Biol Psychiatry April;23(3):533-44) discloses that typical and atypical antipsychotic agents, as a drug class, effectively block the effects of the hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI). DOI is an hallucinogen having high affinity and selectivity as an agonist at 5-HT2A/2C receptors. To identify an antipsychotic compound, the compound is assessed as an antagonist of DOI-induced behaviors in rats. DOI (0.3-10.0 mg/kg; i.p.) produces dose-related behavioral effects including head-and-body shakes, forepaw tapping and skin-jerks. Effects of antipsychotic drugs and other compounds (30 min pretreatment; i.p.) are examined against a fixed dose of DOI (3.0 mg/kg). M100907 (MDL100,907), risperidone, haloperidol, clozapine, iloperidone, olanzapine, amperozide, remoxipride, ritanserin and the neurotensin agonist NT1 (N alpha MeArg-Lys-Pro-Trp-Tle-Leu) antagonize each of the three behavioral effects of DOI. The drawback of this method, however, is that it does not distinguish between typical and atypical anti-psychotic drugs.
Other methods commonly used for identifying potential antipsychotic compounds include prepulse inhibition assays, such as the assays disclosed by Braff et al. (1992, Gating and habituation of the startle reflex in schizophrenic patients. Arch Gen Psychiatry 49:206-215) and by Swerdlow and Geyer (1993, Clozapine and haloperidol in an animal model of sensorimotor gating deficits in schizophrenia. Pharmacol Biochem Behav 44:741-744). Again, the drawback of these method is that they do not distinguish between typical and atypical anti-psychotic drugs. Also, an additional drawback of such behavioral tests is that they are labor intensive.
Surprisingly little is understood, however, about the mechanisms of action of atypical anti-psychotic drugs. Development efforts to develop other atypical anti-psychotic have been hindered by this lack of understanding.
Therefore, there is a need in the art to provide new methods of screening that can be used to develop novel compositions or drugs that can be used to treat psychotic diseases or disorders. Furthermore, there is a need for simple tests of intracellular consequences of antipsychotic action. Since all anti-psychotics act upon multiple receptors, with widely varying downstream effects in terms of both effective relief of symptoms and unwanted side effects, analysis of the intracellular integration of these signals provides a straightforward, cost-effective, and mechanism-based comparison useful for development of the next generation of therapeutic drugs. There is also a need to develop treatments for such diseases or disorders that are due, at least in part, to an aberration or dysregulation of an intracellular signaling pathway regulated by DARPP-32, ERK1, ERK2 and/or CREB. The present invention provides such methods and compositions.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.